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Micro-buddies for macro-resistance

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Rice is the world’s second-most important cereal crop. In 2022-23 alone, 520.4 million metric tons of rice was consumed worldwide. However, its yield is severely threatened by weeds that compete with the crop for resources like nutrients and sunlight. This competition restricts the growth of rice. Out of the around 90 species of weeds growing in rice paddies, the barnyard grass (Echinochloa crus-galli) is the most destructive due to its strong adaptability to the changing environmental conditions and high germination rate. It has developed a strong resistance to a specific type of herbicide called cyhalofop-butyl.

In this study, the authors identified microorganisms on the seed of the weed that were providing resistance to the said weed, investigated the mechanism behind this resistance and figured out a way to counter it in laboratory conditions.

The source of the resistance

The authors first considered if the resistance developed due to a factor internal to the weed, like mutations in the genes the herbicide is targeting, or from an external factor. For that, they studied the herbicide resistance of five successive generations of the weed. They found that the fifth generation was most resistant to the herbicide and that there was no difference between the target genes of the most resistant and the least resistant varieties. The fifth generation also showed the highest bacterial biodiversity of all the generations.

Simpson’s Diversity Index values for the microbiome of the weeds of each generation. A lower value indicates higher biodiversity. **Image source:** Tingting Hu et al., 2023

From this, and some previous studies, the authors proposed that the herbicide resistance was linked to the microorganism biodiversity.

But how does this work?

To figure out the link between the microorganism biodiversity and the herbicide resistance, the microbial species present on the seeds of the herbicide-resistant and herbicide-susceptible weeds were compared. The bacteria Pantoea deleyi was present only on the weed variety exposed to the herbicide. Additionally, it significantly restored the growth of the weed even under herbicide application and showed resistance to cyhalofop-butyl.

The shoot length of the weed, under herbicide application, increased when *Pantoea deleyi* was applied to it. SH-335 is the isolate containing *Pantoea deleyi*. Left column shows shoot length under only herbicide stress, the right column shows shoot length upon *Pantoea deleyi* application. **Image source**: Tingting Hu et al., 2023

What exactly was this microorganism doing to the plant to make it herbicide resistant?

To find that out, the expression of different genes from the resistant weed was analysed and compared to the susceptible one. To understand what that means, we must first understand that each gene codes for a specific protein. If a gene is “expressed” more, it means that more copies of the protein coded by the gene were made, because they are required for a specific function. In this case, the function would be neutralising the herbicide molecules so that they cannot harm the plant anymore, and the plant becomes resistant.

Genes coding for proteins that neutralise herbicide molecules, such as Cytochrome P450 (Cyp P450) and Glutathione S-Transferase (GST), expressed significantly more in the resistant varieties than in the susceptible ones. This confirmed that P. deleyi made the weed herbicide resistant by increasing the expression of genes responsible for herbicide neutralisation.

The expression of genes neutralising herbicide molecules in control (no herbicide *P. deleyi* application), +H (only herbicide application) and +P+H (both herbicide and *P. deleyi* application) conditions. a) expression of Cyp P450 gene, b) expression of GST gene. **Image source:** Tingting Hu et al., 2023

The counter-measure

To top all this, a discovered species of bacteria called Bacillus velezensis was found to potentially inhibit the growth of P. deleyi in lab conditions. This brings exciting prospects for field applications where we could potentially treat the herbicide-resistant weeds with a dose of B. velezensis and make them more susceptible to herbicides.

Various species of bacteria (centre of each image) grown on LB media embedded with *P. deleyi* to assess their ability against it. Image (d) contains *Bacillus velezensis.* Its inhibition capacity is evident from the clearance zone formed around it, indicating that the growth of *P. deleyi* in that zone has been inhibited. **Image source:** Tingting Hu et al., 2023

This approach lays an important foundation to develop more sustainable and eco-friendly solutions for weed management that do not rely on chemical herbicides.

Thus this study brings forward a new mechanism through which weeds may become resistant to herbicides. This knowledge not only gives us a new point of view to understand herbicide resistance, but it also enables us to develop novel methods to counter it by targeting the seed microbiome of the weed.

Link to the original post: Hu, T., Fang, H., Pan, Q., Xu, H., Lv, T., Fan, X., … & Wang, M. (2024). Seed microbiome-mediated herbicide resistance evolution in weeds. The New phytologist, 242(2), 333-343. DOI: https://doi.org/10.1111/nph.19459

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